The present invention relates, first, to DP178 (SEQ ID NO:1), a peptide corresponding to amino acids 638 to 673 of the HIV-1LAI transmembrane protein (TM) gp41, and portions or analogs of DP178 (SEQ ID NO:1), which exhibit anti-membrane fusion capability, antiviral activity, such as the ability to inhibit HIV transmission to uninfected CD-4+ cells, or an ability to modulate intracellular processes involving coiled-coil peptide structures. Further, the invention relates to the use of DP178 (SEQ ID NO:1) and DP178 portions and/or analogs as antifusogenic or antiviral compounds or as inhibitors of intracellular events involving coiled-coil peptide structures. The present invention also relates to peptides analogous to DP107, a peptide corresponding to amino acids 558 to 595 of the HIV-1LAI transmembrane protein (TM) gp41, having amino acid sequences present in other viruses, such as enveloped viruses, and/or other organisms, and further relates to the uses of such peptides. These peptides exhibit anti-membrane fusion capability, antiviral activity, or the ability to modulate intracellular processes involving coiled-coil peptide structures. The present invention additionally relates to methods for identifying compounds that disrupt the interaction between DP178 and DP107, and/or between DP107-like and DP178-like peptides. Further, the invention relates to the use of the peptides of the invention as diagnostic agents. For example, a DP178 peptide may be used as an HIV subtype-specific diagnostic. The invention is demonstrated, first, by way of an Example wherein DP178 (SEQ ID:1), and a peptide whose sequence is homologous to DP178 are each shown to be potent, non-cytotoxic inhibitors of HIV-1 transfer to uninfected CD-4+ cells. The invention is further demonstrated by Examples wherein peptides having structural and/or amino acid motif similarity to DP107 and DP178 are identified in a variety of viral and nonviral organisms, and in examples wherein a number of such identified peptides derived from several different viral systems are demonstrated to exhibit antiviral activity.
Membrane fusion is a ubiquitous cell biological process (for a review, see White, J. M., 1992, Science 258:917-924). Fusion events which mediate cellular housekeeping functions, such as endocytosis, constitutive secretion, and recycling of membrane components, occur continuously in all eukaryotic cells.
Additional fusion events occur in specialized cells. Intracellularly, for example, fusion events are involved in such processes as occur in regulated exocytosis of hormones, enzymes and neurotransmitters. Intercellularly, such fusion events feature prominently in, for example, sperm-egg fusion and myoblast fusion.
Fusion events are also associated with disease states. For example, fusion events are involved in the formation of giant cells during inflammatory reactions, the entry of all enveloped viruses into cells, and, in the case of human immunodeficiency virus (HIV), for example, are responsible for the virally induced cell-cell fusion which leads to cell death.
The human immunodeficiency virus (HIV) has been implicated as the primary cause of the slowly degenerative immune system disease termed acquired immune deficiency syndrome (AIDS) (Barre-Sinoussi, F. et al., 1983, Science 220:868-870; Gallo, R. et al., 1984, Science 224:500-503). There are at least two distinct types of HIV: HIV-1 (Barre-Sinoussi, F. et al., 1983, Science 220:868-870; Gallo R. et al., 1984, Science 224:500-503) and HIV-2 (Clavel, F. et al., 1986, Science 233:343-346; Guyader, M. et al., 1987, Nature 326:662-669). Further, a large amount of genetic heterogeneity exists within populations of each of these types. Infection of human CD-4+ T-lymphocytes with an HIV virus leads to depletion of the cell type and eventually to opportunistic infections, neurological dysfunctions, neoplastic growth, and ultimately death.
HIV is a member of the lentivirus family of retroviruses (Teich, N. et al., 1984, RNA Tumor Viruses, Weiss, R. et al., eds., CSH-Press, pp. 949-956). Retroviruses are small enveloped viruses that contain a diploid, single-stranded RNA genome, and replicate via a DNA intermediate produced by a virally-encoded reverse transcriptase, an RNA-dependent DNA polymerase (Varmus, H., 1988, Science 240:1427-1439). Other retroviruses include, for example, oncogenic viruses such as human T-cell leukemia viruses (HTLV-I,-II,-III), and feline leukemia virus.
The HIV viral particle consists of a viral core, composed of capsid proteins, that contains the viral RNA genome and those enzymes required for early replicative events. Myristylated Gag protein forms an outer viral shell around the viral core, which is, in turn, surrounded by a lipid membrane enveloped derived from the infected cell membrane. The HIV enveloped surface glycoproteins are synthesized as a single 160 Kd precursor protein which is cleaved by a cellular protease during viral budding into two glycoproteins, gp41 and gp120. gp41 is a transmembrane protein and gp120 is an extracellular protein which remains non-covalently associated with gp41, possibly in a trimeric or multimeric form (Hammarskjold, M. and Rekosh, D., 1989, Biochem. Biophys. Acta 989:269-280).
HIV is targeted to CD-4+ cells because the CD-4 cell surface protein acts as the cellular receptor for the HIV-1 virus (Dalgleish, A. et al., 1984, Nature 312:763-767; Klatzmann et al., 1984, Nature 312:767-768; Maddon et al., 1986, Cell 47:333-348). Viral entry into cells is dependent upon gpl2O binding the cellular CD-4+ receptor molecules (McDougal, J. S. et al., 1986, Science 231:382-385; Maddon, P. J. et al., 1986, Cell 47:333-348) and thus explains HIV""s tropism for CD-4+ cells, while gp41 anchors the enveloped glycoprotein complex in the viral membrane.
HIV infection is pandemic and HIV associated diseases represent a major world health problem. Although considerable effort is being put into the successful design of effective therapeutics, currently no curative anti-retroviral drugs against AIDS exist. In attempts to develop such drugs, several stages of the HIV life cycle have been considered as targets for therapeutic intervention (Mitsuya, H. et al., 1991, FASEB J. 5:2369-2381). For example, virally encoded reverse transcriptase has been one focus of drug development. A number of reverse-transcriptase-targeted drugs, including 2xe2x80x2,3xe2x80x2-dideoxynucleoside analogs such as AZT, ddI, ddC, and d4T have been developed which have been shown to been active against HIV (Mitsuya, H. et al., 1991, Science 249:1533-1544). While beneficial, these nucleoside analogs are not curative, probably due to the rapid appearance of drug resistant HIV mutants (Lander, B. et al., 1989, Science 243:1731-1734). In addition, the drugs often exhibit toxic side effects such as bone marrow suppression, vomiting, and liver function abnormalities.
Attempts are also being made to develop drugs which can inhibit viral entry into the cell, the earliest stage of HIV infection. Here, the focus has thus far been on CD4, the cell surface receptor for HIV. Recombinant soluble CD4, for example, has been shown to inhibit infection of CD-4+ T-cells by some HIV-1 strains (Smith, D. H. et al., 1987, Science 238:1704-1707). Certain primary HIV-1 isolates, however, are relatively less sensitive to inhibition by recombinant CD-4 (Daar, E. et al., 1990, Proc. Natl. Acad. Sci. USA 87:6574-6579). In addition, recombinant soluble CD-4 clinical trials have produced inconclusive results (Schooley, R. et al., 1990, Ann. Int. Med. 112:247-253; Kahn, J. O. et al., 1990, Ann. Int. Med. 112:254-261; Yarchoan, R. et al., 1989, Proc. Vth Int. Conf. on AIDS, p. 564, MCP 137).
The late stages of HIV replication, which involve crucial virus-specific secondary processing of certain iral proteins, have also been suggested as possible anti-HIV drug targets. Late stage processing is dependent on the activity of a viral protease, and drugs are being developed which inhibit this protease (Erickson, J., 1990, Science 249:527-533). The clinical outcome of these candidate drugs is still in question.
Attention is also being given to the development of vaccines for the treatment of HIV infection. The HIV-1 enveloped proteins (gp160, gp120, gp41) have been shown to be the major antigens for anti-HIV antibodies present in AIDS patients (Barin, et al., 1985, Science 228:1094-1096). Thus far, therefore, these proteins seem to be the most promising candidates to act as antigens for anti-HIV vaccine development. To this end, several groups have begun to use various portions of gp160, gp120, and/or gp41 as immunogenic targets for the host immune system. See for example, Ivanoff, L. et al., U.S. Pat. No. 5,141,867; Saith, G. et al., WO 92/22,654; Shafferman, A., WO 91/09,872; Formoso, C. et al., WO 90/07,119. Clinical results concerning these candidate vaccines, however, still remain far in the future.
Thus, although a great deal of effort is being directed to the design and testing of anti-retroviral drugs, a truly effective, non-toxic treatment is still needed.
The present invention relates, first, to DP178 (SEQ ID:1), a 36-amino acid synthetic peptide corresponding to amino acids 638 to 673 of the transmembrane protein (TM) gp41 from the HIV-1 isolate LAI (HIV-1LAI), which exhibits potent anti-HIV-1 activity. As evidenced by the Example presented below, in Section 6, the DP178 (SEQ ID:1) antiviral activity is so high that, on a weight basis, no other known anti-HIV agent is effective at concentrations as low as those at which DP178 (SEQ ID:1) exhibits its inhibitory effects.
The invention further relates to those portions and analogs of DP178 which also show such antiviral activity, and/or show anti-membrane fusion capability, or an ability to modulate intracellular processes involving coiled-coil peptide structures. The term xe2x80x9cDP178 analogxe2x80x9d refers to a peptide which contains an amino acid sequence corresponding to the DP178 peptide sequence present within the gp41 protein of HIV-1LAI, but found in viruses and/or organisms other than HIV-1LAI. Such DP178 analog peptides may, therefore, correspond to DP178-like amino acid sequences present in other viruses, such as, for example, enveloped viruses, such as retroviruses other than HIV-1LAI, as well as non-enveloped viruses. Further, such analogous DP178 peptides may also correspond to DP178-like amino acid sequences present in nonviral organisms.
The invention further relates to DP107 (SEQ ID NO:89) peptide analogs. DP107 is a peptide corresponding to amino acids 558-595 of the HIV-1LAI transmembrane protein (TM) gp41. The term xe2x80x9cDP107 analogxe2x80x9d as used herein refers to a peptide which contains an amino acid sequence corresponding to the DP107 peptide sequence present within the gp41 protein of HIV-1LAI, but found in viruses and organisms other than HIV-1LAI. Such DP107 analog peptides may, therefore, correspond to DP107-like amino acid sequences present in other viruses, such as, for example, enveloped viruses, such as retroviruses other than HIV-1LAI, as well as non-enveloped viruses. Further, such DP107 analog peptides may also correspond to DP107-like amino acid sequences present in nonviral organisms.
Further, the peptides of the invention include DP107 analog and DP178 analog peptides having amino acid sequences recognized or identified by the 107xc3x97178xc3x974, ALLMOTI5 and/or PLZIP search motifs described herein.
The peptides of the invention may, for example, exhibit antifusogenic activity, antiviral activity, and/or may have the ability to modulate intracellular processes which involve coiled-coil peptide structures. With respect to the antiviral activity of the peptides of the invention, such an antiviral activity includes, but is not limited to the inhibition of HIV transmission to uninfected CD-4+ cells. Additionally, the antifusogenic capability, antiviral activity or intracellular modulatory activity of the peptides of the invention merely requires the presence of the peptides of the invention, and, specifically, does not require the stimulation of a host immune response directed against such peptides.
The peptides of the invention may be used, for example, as inhibitors of membrane fusion-asociated events, such as, for example, the inhibition of human and non-human retroviral, especially HIV, transmission to uninfected cells. It is further contemplated that the peptides of the invention may be used as modulators of intracellular events involving coiled-coil peptide structures.
The peptides of the invention may, alternatively, be used to identify compounds which may themselves exhibit antifusogenic, antiviral, or intracellular modulatory activity. Additional uses include, for example, the use of the peptides of the invention as organism or viral type and/or subtype-specific diagnostic tools.
The terms xe2x80x9cantifusogenicxe2x80x9d and xe2x80x9canti-membrane fusionxe2x80x9d, as used herein, refer to an agent""s ability to inhibit or reduce the level of membrane fusion events between two or more moieties relative to the level of membrane fusion which occurs between said moieties in the absence of the peptide. The moieties may be, for example, cell membranes or viral structures, such as viral envelopes or pili. The term xe2x80x9cantiviralxe2x80x9d, as used herein, refers to the compound""s ability to inhibit viral infection of cells, via, for example, cell-cell fusion or free virus infection. Such infection may involve membrane fusion, as occurs in the case of enveloped viruses, or some other fusion event involving a viral structure and a cellular structure (e.g., such as the fusion of a viral pilus and bacterial membrane during bacterial conjugation).
It is also contemplated that the peptides of the invention may exhibit the ability to modulate intracellular events involving coiled-coil peptide structures. xe2x80x9cModulatexe2x80x9d, as used herein, refers to a stimulatory or inhibitory effect on the intracellular process of interest relative to the level or activity of such a process in the absence of a peptide of the invention.
Embodiments of the invention are demonstrated below wherein an extremely low concentration of DP178 (SEQ ID:1), and very low concentrations of a DP178 homolog (SEQ ID:3) are shown to be potent inhibitors of HIV-1 mediated CD-4+ cell-cell fusion (i.e., syncytial formation) and infection of CD-4+ cells by cell-free virus. Further, it is shown that DP178 (SEQ ID:1) is not toxic to cells, even at concentrations 3 logs higher than the inhibitory DP-178 (SEQ ID:1) concentration.
The present invention is based, in part, on the surprising discovery that the DP107 and DP178 domains of the HIV gp41 protein non-covalently complex with each other, and that their interaction is required for the normal infectivity of the virus. This discovery is described in the Example presented, below, in Section 8. The invention, therefore, further relates to methods for identifying antifusogenic, including antiviral, compounds that disrupt the interaction between DP107 and DP178, and/or between DP107-like and DP178-like peptides.
Additional embodiments of the invention (specifically, the Examples presents in Sections 9-16 and 19-25, below) are demonstrated, below, wherein peptides, from a variety of viral and nonviral sources, having structural and/or amino acid motif similarity to DP107 and DP178 are identified, and search motifs for their identification are described. Further, Examples (in Sections 17, 18, 25-29) are presented wherein a number of the peptides of the invention are demonstrated exhibit substantial antiviral activity or activity predictive of antiviral activity.
Peptides are defined herein as organic compounds comprising two or more amino acids covalently joined by peptide bonds. Peptides may be referred to with respect to the number of constituent amino acids, i.e., a dipeptide contains two amino acid residues, a tripeptide contains three, etc. Peptides containing ten or fewer amino acids may be referred to as oligopeptides, while those with more than ten amino acid residues are polypeptides. Such peptides may also include any of the modifications and additional amino and carboxy groups as are described herein.
Peptide sequences defined herein are represented by one-letter symbols for amino acid residues as follows:
FIG. 1. Amino acid sequence of DP178 (SEQ ID:1) derived from HIVLAI; DP178 homologs derived from HIV-1SF2 (DP-185; SEQ ID:3), HIV-1RF (SEQ ID:4), and HIV-1MN (SEQ ID:5); DP178 homologs derived from amino acid sequences of two prototypic HIV-2 isolates, namely, HIV-2rod (SEQ ID:6) and HIV-2NIHZ (SEQ ID:7); control peptides: DP-180 (SEQ ID:2), a peptide incorporating the amino acid residues of DP178 in a scrambled sequence; DP-118 (SEQ ID:10) unrelated to DP178, which inhibits HIV-1 cell free virus infection; DP-125 (SEQ ID:8), unrelated to DP178, also inhibits HIV-1 cell free virus infection; DP-116 (SEQ ID:9), unrelated to DP178, is negative for inhibition of HIV-1 infection when tested using a cell-free virus infection assay. Throughout the figures, the one letter amino acid code is used.
FIG. 2. Inhibition of HIV-1 cell-free virus infection by synthetic peptides. IC50 refers to the concentration of peptide that inhibits RT production from infected cells by 50% compared to the untreated control. Control: the level of RT produced by untreated cell cultures infected with the same level of virus as treated cultures.
FIG. 3. Inhibition of HIV-1 and HIV-2 cell-free virus infection by the synthetic peptide DP178 (SEQ ID:1). IC50: concentration of peptide that inhibits RT production by 50% compared to the untreated control. Control: Level of RT produced by untreated cell cultures infected with the same level of virus as treated cultures.
FIGS. 4A-4B. Fusion Inhibition Assays. FIG. 4A: DP178 (SEQ ID:1) inhibition of HIV-1 prototypic isolate-mediated syncytial formation; data represents the number of virus-induced syncytial per cell. FIG. 4B: DP-180 (SEQ ID:2) represents a scrambled control peptide; DP-185 (SEQ ID:3) represents a DP178 homolog derived from HIV-1SF2 isolate; Control, refers to the number of syncytial produced in the absence of peptide.
FIG. 5. Fusion inhibition assay: HIV-1 vs. HIV-2. Data represents the number of virus-induced syncytial per well. ND: not done.
FIG. 6. Cytotoxicity study of DP178 (SEQ ID:1) and DP-116 (SEQ ID:9) on CEM cells. Cell proliferation data is shown.
FIG. 7. Schematic representation of HIV-gp41 and maltose binding protein (MBP)-gp41 fusion proteins. DP107 and DP178 are synthetic peptides based on the two putative helices of gp41. The letter P in the DP107 boxes denotes an Ile to Pro mutation at amino acid number 578. Amino acid residues are numbered according to Meyers et al., xe2x80x9cHuman Retroviruses and AIDSxe2x80x9d, 1991, Theoret. Biol. and Biophys. Group, Los Alamos Natl. Lab., Los Alamos, N.Mex. The proteins are more fully described, below, in Section 8.1.1.
FIG. 8. A point mutation alters the conformation and anti-HIV activity of M41.
FIG. 9. Abrogation of DP178 anti-HIV activity. Cell fusion assays were carried out in the presence of 10 nM DP178 and various concentrations of M41xcex94178 or M41Pxcex94178.
FIG. 10. Binding of DP178 to leucine zipper of gp41 analyzed by FAb-D ELISA.
FIGS. 11A-B. Models for a structural transition in the HIV-1 TM protein. Two models are proposed which indicate a structural transition from a native oligomer to a fusogenic state following a trigger event (possibly gp120 binding to CD4). Common features of both models include (1) the native state is held together by noncovalent protein-protein interactions to form the heterodimer of gp120/41 and other interactions, principally though gp41 interactive sites, to form homo-oligomers on the virus surface of the gp120/41 complexes; (2) shielding of the hydrophobic fusogenic peptide at the N-terminus (F) in the native state; and (3) the leucine zipper domain (DP107) exists as a homo-oligomer coiled coil only in the fusogenic state. The major differences in the two models include the structural state (native or fusogenic) in which the DP107 and DP178 domains are complexed to each other. In the first model (FIG. 11A) this interaction occurs in the native state and in the second (FIG. 11B), it occurs during the fusogenic state. When triggered, the fusion complex in the model depicted in (A) is generated through formation of coiled-coil interactions in homologous DP107 domains resulting in an extended xcex1-helix. This conformational change positions the fusion peptide for interaction with the cell membrane. In the second model (FIG. 11B), the fusogenic complex is stabilized by the association of the DP178 domain with the DP107 coiled-coil.
FIG. 12. Motif design using heptad repeat positioning of amino acids of known coiled-coils [GCN4:(SEQ ID NO:84); C-FOS:(SEQ ID NO:85); C-JUN: (SEQ ID NO:86); C-MYC:(SEQ ID NO:87); FLU LOOP 36:(SEQ ID NO:88)].
FIG. 13. Motif design using proposed heptad repeat positioning of amino acids of DP107 and DP178.
FIG. 14. Hybrid motif design crossing GCN4 and DP107.
FIG. 15. Hybrid motif design crossing GCN4 and DP178.
FIG. 16. Hybrid motif design 107xc3x97178xc3x974, crossing DP107 (SEQ ID NO:89) and DP178 (SEQ ID NO:1). This motif was found to be the most consistent at identifying relevant DP107-like and DP178-like peptide regions.
FIG. 17. Hybrid motif design crossing GCN4, DP107, and DP178.
FIG. 18. Hybrid motif design ALLMOTI5 crossing GCN4, DP107, DP178, c-Fos c-Jun, c-Myc, and Flu Loop 36.
FIG. 19. PLZIP motifs designed to identify N-terminal proline-leucine zipper motifs.
FIG. 20. Search results for HIV-1 (BRU isolate) enveloped protein gp41 (SEQ ID NO:90). Sequence search motif designations: Spades (): 107xc3x97178xc3x974; Hearts (♥) ALLMOTI5; Clubs (): PLZIP; Diamonds (♦): transmembrane region (the putative transmembrane domains were identified using a PC/Gene program designed to search for such peptide regions). Asterisk (*): Lupas method. The amino acid sequences identified by each motif are bracketed by the respective characters. Representative sequences chosen based on 107xc3x97178xc3x974 searches are underlined and in bold. DP107 and DP178 sequences are marked, and additionally double-underlined and italicized.
FIG. 21. Search results for human respiratory syncytial virus (RSV) strain A2 fusion glycoprotein F1 (SEQ ID NO:91). Sequence search motif designations are as in FIG. 20.
FIG. 22. Search results for simian immunodeficiency virus (SIV) enveloped protein gp41 (AGM3 isolate) (SEQ ID NO:92). Sequence search motif designations are as in FIG. 20.
FIG. 23. Search results for canine distemper virus (strain Onderstepoort) fusion glycoprotein 1 (SEQ ID NO:93). Sequence search motif designations are as in FIG. 20.
FIG. 24. Search results for newcastle disease virus (strain Australia-Victoria/32) fusion glycoprotein F1 (SEQ ID NO:94). Sequence search motif designations are as in FIG. 20.
FIG. 25. Search results for human parainfluenza 3 virus (strain NIH 47885) fusion glycoprotein F1 (SEQ ID NO:95). Sequence search motif designations are as in FIG. 20.
FIG. 26. Search results for influenza A virus (strain A/AICHI/2/68) hemagglutinin precursor HA2 (SEQ ID NO:96). Sequence search designations are as in FIG. 20.
FIGS. 27A-F: Respiratory Syncytial Virus (RSV) peptide (SEQ ID NO:97) antiviral and circular dichroism data. FIGS. 27A-C: Peptides derived from the F2 DP178/DP107-like region: [T-22: (SEQ ID NO:121); T-68: (SEQ ID NO:122); T-334: (SEQ ID NO:123); T-371: (SEQ ID NO:124); T-372: (SEQ ID NO:125); T-373: (SEQ ID NO:126); T-374: (SEQ ID NO:127); T-375: (SEQ ID NO:128); T-575: (SEQ ID NO:129)]. Antiviral and CD data. FIGS. 27D-F: Peptides derived from the F1 DP107-like region: [F1-107: (SEQ ID NO:98); T-12: (SEQ ID NO:130); T-13: (SEQ ID NO:131); T-15: (SEQ ID NO:132); T-9: (SEQ ID NO:133); T-28: (SEQ ID NO:134); T-30: (SEQ ID NO:135); T-66: (SEQ ID NO:136); T-576: (SEQ ID NO:137)]. Peptide and CD data.
Antiviral activity (AV) is represented by the following qualitative symbols:
xe2x80x9cxe2x88x92xe2x80x9d, negative antiviral activity;
xe2x80x9c+/xe2x88x92xe2x80x9d antiviral activity at greater than 100 xcexcg/ml;
xe2x80x9c+xe2x80x9d, antiviral activity at between 50-100 xcexcg/ml;
xe2x80x9c++xe2x80x9d, antiviral activity at between 20-50 xcexcg/ml;
xe2x80x9c+++xe2x80x9d, antiviral activity at between 1-20 xcexcg/ml;
xe2x80x9c++++xe2x80x9d, antiviral activity at  less than 1 xcexcg/ml.
CD data, referring to the level of helicity is represented by the following qualitative symbol:
xe2x80x9cxe2x88x92xe2x80x9d, no helicity;
xe2x80x9c+xe2x80x9d, 25-50% helicity;
xe2x80x9c++xe2x80x9d, 50-75% helicity;
xe2x80x9c+++xe2x80x9d, 75-100% helicity.
IC50 refers to the concentration of peptide necessary to produce only 50% of the number of syncytial relative to infected control cultures containing no peptide. IC50 values were obtained using purified peptides only.
FIGS. 28A-C: Respiratory Syncytial Virus (RSV) DP178-like region (F1) peptide antiviral and CD data [F1-178: (SEQ ID NO:99); T-71: (SEQ ID NO:138); T-384: (SEQ ID NO:139); T-616: (SEQ ID NO:140); T-617: (SEQ ID NO:141); T-662: (SEQ ID NO:142); T-665: (SEQ ID NO:143); T-671: (SEQ ID NO:144); T-730: (SEQ ID NO:145)]. Antiviral symbols, CD symbols, and IC50 are as in FIGS. 27A-F. IC50 values were obtained using purified peptides only.
FIGS. 29A-E. Peptides derived from the HPIV3 F1 DP107-like region. Peptide antiviral and CD data [HPFI 107: (SEQ ID NO:100); T-42: (SEQ ID NO:146); T-39: (SEQ ID NO:147); T-40: (SEQ ID NO:148); T-45: (SEQ ID NO:149); T-46: (SEQ ID NO:150); T-582: (SEQ ID NO:151)]. Antiviral symbols, CD symbols, and IC50 are as in FIGS. 27A-F. Purified peptides were used to obtain IC50 values, except where the values are marked by an asterisk (*); in such cases, the IC50 values were obtained using a crude peptide preparation.
FIGS. 30A-C. Peptides derived from the HPIV3 F1 DP178-like region. Peptide antiviral and CD data [HPF3178: (SEQ ID NO:101); T-269: (SEQ ID NO:152); T-626: (SEQ ID NO:153); T-383: (SEQ ID NO:154); T-577: (SEQ ID NO:155); T-578: (SEQ ID NO:156); T-579: (SEQ ID NO:157)]. Antiviral symbols, CD symbols, and IC50 are as in FIGS. 27A-F. Purified peptides were used to obtain IC50 values, except where the values are marked by an asterisk (*); in such cases, the IC50 values were obtained using a crude peptide preparation.
FIG. 31. Motif search results for simian immunodeficiency virus (SIV) isolate MM251, enveloped polyprotein gp41 (SEQ ID NO:102). Sequence search designations are as in FIG. 20.
FIG. 32. Motif search results for Epstein-Barr Virus (Strain B95-8), glycoprotein gp110 precursor (designated gp115). BALF4 (SEQ ID NO:103): Sequence search designations are as in FIG. 20.
FIG. 33. Motif search results for Epstein-Barr Virus (Strain B95-8), BZLF1 trans-activator protein (designated EB1 or Zebra) (SEQ ID NO:104). Sequence search designations are as in FIG. 20. Additionally, xe2x80x9c@xe2x80x9d refers to a well known DNA binding domain and xe2x80x9c+xe2x80x9d refers to a well known dimerization domain, as defined by Flemington and Speck (Flemington, E. and Speck, S. H., 1990, Proc. Natl. Acad. Sci. USA 87:9459-9463).
FIG. 34. Motif search results for measles virus (strain Edmonston), fusion glycoprotein F1 (SEQ ID NO:105). Sequence search designations are as in FIG. 20.
FIG. 35. Motif search results for Hepatitis B Virus (subtype AYW), major surface antigen precursor S. (SEQ ID NO:106) Sequence search designations are as in FIG. 20.
FIG. 36. Motif search results for simian Mason-Pfizer monkey virus, enveloped (TM) protein gp20 (SEQ ID NO:107). Sequence search designations are as in FIG. 20.
FIG. 37. Motif search results for Pseudomonas aerginosa, fimbrial protein (SEQ ID NO:110) (Pilin) (SEQ ID NO:108). Sequence search designations are as in FIG. 20.
FIG. 38. Motif search results for Neisseria gonorrhoeae fimbrial protein (Pilin) (SEQ ID NO:109). Sequence search designations are as in FIG. 20.
FIG. 39. Motif search results for Hemophilus influenzae fimbrial protein. Sequence search designations are as in FIG. 20.
FIG. 40. Motif search results for Staphylococcus aureus, toxic shock syndrome toxin-1 (SEQ ID NO:111). Sequence search designations are as in FIG. 20.
FIG. 41. Motif search results for Staphylococcus aureus enterotoxin Type E (SEQ ID NO:112). Sequence search designations are as in FIG. 20.
FIG. 42. Motif search results for Staphylococcus aureus enterotoxin A (SEQ ID NO:113). Sequence search designations are as in FIG. 20.
FIG. 43. Motif search results for Escherichia coli, heat labile enterotoxin A (SEQ ID NO:114). Sequence search designations are as in FIG. 20.
FIG. 44. Motif search results for human c-fos proto-oncoprotein (SEQ ID NO:115). Sequence search designations are as in FIG. 20.
FIG. 45. Motif search results for human lupus KU autoantigen protein P70 (SEQ ID NO:116). Sequence search designations are as in FIG. 20.
FIG. 46. Motif search results for human zinc finger protein 10 (SEQ ID NO:117). Sequence search designations are as in FIG. 20.
FIGS. 47A-B: Measles virus (MeV) fusion protein DP178-like region antiviral and CD data [T-252AO: (SEQ ID NO:118); T-268AO: (SEQ ID NO:119)]. Antiviral symbols, CD symbols, and IC50 are as in FIGS. 27A-F.
FIGS. 48A-B: Simian immunodeficiency virus (SIV) TM (fusion) protein DP178-like region antiviral data (SEQ ID NO:120). Antiviral symbols are as in FIGS. 27A-F. xe2x80x9cNTxe2x80x9d, not tested.
FIGS. 49A-L: DP178-derived peptide antiviral data [T50: (SEQ ID NO:159); T234: (SEQ ID NO:161); T235: (SEQ ID NO:162); T570: (SEQ ID NO:163); T381: (SEQ ID NO:164); T677: (SEQ ID NO:165); T589: (SEQ ID NO:166); T590: (SEQ ID NO:167); T591: (SEQ ID NO:168); T270: (SEQ ID NO:169); T271: (SEQ ID NO:170); T273: (SEQ ID NO:171); T608: (SEQ ID NO:172); T609: (SEQ ID NO:173); T610: (SEQ ID NO:174); T611: (SEQ ID NO:175); T612: (SEQ ID NO:176); T595: (SEQ ID NO:177); T95: (SEQ ID NO:178); T96: (SEQ ID NO:179); T97: (SEQ ID NO:180); T98: (SEQ ID NO:181); T99: (SEQ ID NO:182); T103: (SEQ ID NO:183); T212: (SEQ ID NO:184); T213: (SEQ ID NO:185); T214: (SEQ ID NO:186); T215: (SEQ ID NO:187); T216: (SEQ ID NO:188); T229: (SEQ ID NO:189); T230: (SEQ ID NO:-190); T231: (SEQ ID NO:191); T379: (SEQ ID NO:192); T701: (SEQ ID NO:193); T702: (SEQ ID NO:194); T703: (SEQ ID NO:195); T704: (SEQ ID NO:196); T705: (SEQ ID NO:197); T706: (SEQ ID NO:198); T156: (SEQ ID NO:199); T90: (SEQ ID NO:200)].
The peptides listed herein were derived from the region surrounding the HIV-1 BRU isolate DP178 region (e.g., gp41 amino acid residues 615-717).
In instances where peptides contained DP178 point mutations, the mutated amino acid residues are shown with a shaded background. In instances in which the test peptide has had an amino and/or carboxy-terminal group added or removed (apart from the standard amido- and acetyl-blocking groups found on such peptides), such modifications are indicated. FIGS. 49A, 49C: The column to the immediate right of the name of the test peptide indicates the size of the test peptide and points out whether the peptide is derived from a one amino acid peptide xe2x80x9cwalkxe2x80x9d across the DP178 region. The next column to the right indicates whether the test peptide contains a point mutation, while the column to its right indicates whether certain amino acid residues have been added to or removed from the DP178-derived amino acid sequence. FIGS. 49E, 49G: The column to the immediate right of the test peptide name indicates whether the peptide represents a DP178 truncation, the next column to the right points out whether the peptide contains a point mutation, and the column to its right indicates whether the peptide contains amino acids which have been added to or removed from the DP178 sequence itself. FIGS. 49I, 49K: The column to the immediate right of the test peptide name indicates whether the test peptide contains a point mutation, while the column to its right is indicates whether amino acid residues have been added to or removed from the DP178 sequence itself. IC50 is as defined in FIGS. 27A-F, and IC50 values were obtained using purified peptides except where marked with an asterisk (*), in which case the IC50 was obtained using a crude peptide preparation.
FIGS. 50A-B: DP107 and DP107 gp41 region truncated peptide antiviral data (SEQ ID NO:201). IC50 as defined in FIGS. 27A-F, and IC50 values were obtained using purified peptides except where marked with an asterisk (*), in which case the IC50 was obtained using a crude peptide preparation.
FIGS. 51A-C: Epstein-Barr virus Strain B95-8 BZLF1 DP178/DP107 analog region peptide walks and electrophoretic mobility shift assay results. The peptides [173-219: (SEQ ID NO:202); 185-230: (SEQ ID NO:203); T-446: (SEQ ID NO:204); 197-242: (SEQ ID NO:205); T-458: (SEQ ID NO:206); 209-246: (SEQ ID NO:207)] (T-423 to T-434, FIG. 51A; T-435 to T-466, FIG. 51B; T-447 to T-449, T-451 to T-458 and T-459 to T-461, FIG. 51C) represent one amino acid residue xe2x80x9cwalksxe2x80x9d through the EBV Zebra protein region from amino acid residue 173 to 246.
The amino acid residue within this region which corresponds to the first amino acid residue of each peptide is listed to the left of each peptide, while the amino acid residue within this region which corresponds to the last amino acid residue of each peptide is listed to the right of each peptide. The length of each test peptide is listed at the far right of each line, under the heading xe2x80x9cResxe2x80x9d.
xe2x80x9cACTxe2x80x9d refers to a test peptide""s ability to inhibit Zebra binding to its response element. xe2x80x9c+xe2x80x9d refers to a visible, but incomplete, abrogation of the response element/Zebra homodimer complex; xe2x80x9c+++xe2x80x9d refers to a complete abrogation of the complex; and xe2x80x9cxe2x88x92xe2x80x9d represents a lack of complex disruption.
FIGS. 52A-B. Hepatitis B virus subtype AYW major surface antigen precursor S protein DP178/DP107 analog region and peptide walks. 52A depicts Domain I (SEQ ID NO:208) (S protein amino acid residues 174-219), which contains a potential DP178/DP107 analog region. In addition, peptides are listed which represent one amino acid peptide xe2x80x9cwalksxe2x80x9d through domain I. 52B depicts Domain II (SEQ ID NO:209) (S protein amino acid residues 233-290), which contains a second potential DP178/DP107 analog region. In addition, peptides are listed which represent one amino acid peptide xe2x80x9cwalksxe2x80x9d through domain II.